The von Willebrand factor is a multimetric blood protein with molecular weights within of about 200 kDa and of about 20 000 kDa, and even more. This protein, that is synthetised by blood platelets and endothelial cells, plays a key role in the struggle against bleeding insofar as the FvW acts as a gelifying plug which spreads over a vascular breach providing for the adhesion of platelets in order to carry out the first phase of the hemostasis, namely the formation of a “platelet-thrombus” (thrombus). The coagulation phenomena, intended to consolidate the arrest of the bleeding by forming an insoluble fibrin clot, will take place around this thrombus. In the blood circulation, the FvW ensures also the stabilization and the transport of Factor VIII with which it is associated in complexes of various sizes, said Factor VIII being thus protected against a rapid degradation by proteolysis because of the sensitivity of the isolated Factor VIII to proteases.
A congenital deficiency of FvW or genetic mutations, that modify the properties of FvW, causes the Willebrand disease which is indicated by troubles of primary hemostasis and of blood clotting.
The availability of very high purity human plasma derivatives enriched by FvW, suitable to multiple and repeated injections, is therefore of utmost importance in the treatment of this disease. Indeed, samples of FvW with insufficient purity, obtained by fractionation of human plasma, contain various contaminants (residual proteins) capable to induce undesirable immunological reactions. Moreover, the administration of von Willebrand factor associated with Factor VIII may cause the risk of thrombosis or hypercoagulability in the treated patient (Makris et al, Thromb. Haemost. 88, 2002, pp. 377-378, Manucci P. M., Thromb. Haemost. 88, 2002, pp. 378-379).
Various processes for the preparation of FvW concentrates are typically associating steps of precipitation of a plasma fraction intended to remove the major part of undesirable proteins (fibrinogen, fibronectin, etc.), and/or chromatographies (ion exchange, affinity, immunoaffinity, size exclusion, etc.) aiming at obtaining very high purity concentrates, exhibiting a high specific activity and permitting to preserve the integrity of multimetric forms, especially those of high molecular weight, which are of utmost biological importance in the healing processes.
By way of example, reference can be made to the patent EP 0 359 593 which discloses the separation of proteins of a cryoprecipitated fraction of plasma implementing several steps of anion exchange chromatography leading to the purification of FvW.
The patent EP 0 503 991 discloses a process for preparing an industrial-scale FvW concentrate including a pre-purification step of a cryoprecipitated fraction of plasma and three successive chromatography steps, the third one being an affinity chromatography on a column of gelatine immobilized on agarose. The thus obtained FvW concentrate exhibits a specific activity higher than 100 UI RCo/mg expressed in ristocetine cofactor activity units per mg of proteins and a content of high molecular weight multimers comparable with that in the starting plasma.
The Patent Application EP 0 934 748 describes a process for the preparation of FvW including the combination of anion exchange and cation exchange chromatographies. The obtained FvW fractions exhibit a specific activity higher than 100 IU FvW:Ag/mg expressed in FvW antigen units per mg of protein, but still contain notable proportions of Factor VIII.
The patent U.S. Pat. No. 6,579,723 describes a process for preparing a highly purified FvW by immunoaffinity chromatography wherein the immunoadsorbents are anti-FvW antibodies. An additional step of purification by affinity chromatography on heparin can also be provided. However, the drawback of the purification by immunoaffinity is the possible presence of residual antibodies that can lead to immunological reactions.
The patent EP 0 383 234 teaches the preparation of a pasteurised FvW concentrate by means of an anion exchange chromatography, involving acidic solutions (pH of 5.5 to 6.5) containing carbohydrates, in order to fix the Factor VIII on the anion exchanger. The joint recovery of not-retained FvW, fibronectin and fibrinogen by washing of the support requires further precipitation steps in order to isolate a purified FvW concentrate.
The drawback of the above described processes lies in that they require several successive steps, especially chromatographic steps, causing problems related to the yield and to the clumsiness of the industrial-scale implementation. The processes can use, depending on the case, chromatographic supports with ligands of animal origin, such as based on gelatine, on heparin or on collagen, capable of acting as vectors of pathogenic prions responsible of spongiform encephalopathy, or of other viruses of the considered animal species. These difficulties are even amplified owing to the necessity to include in the process a virus inactivation treatment and, if need be, additional steps for the removal of virucide agents. Besides, the FvW concentrates obtained by these processes are not free from Factor VIII that represents the drawback of risk of thrombosis for the patients. Furthermore, the cleaning or washing of the affinity supports and their sanitation as well are difficult because of the fragility of the ligand, that prevents the use of cleaning solutions with a strong disinfectant character (sodium hypochlorite, soda, potassium hydroxide, etc.). Finally, the affinity supports have rather short lifetimes and their frequent replacement represents relatively high expenses, that are a burden to the cost price of the treated product.
Considering the continuously growing needs of high purity FvW fractions, the Applicant attempted to develop a new process for the preparation of FvW, which can be implemented by very simple means, giving a high yield, without the need to use chromatographic supports with ligands of animal origin and leading to a standardized high purity FvW concentrate endowed with a high specific activity and free from Factor VIII.